scholarly journals Opening a Novel Biosynthetic Pathway to Dihydroxyacetone and Glycerol in Escherichia coli Mutants through Expression of a Gene Variant (fsaAA129S) for Fructose 6-Phosphate Aldolase

2020 ◽  
Vol 21 (24) ◽  
pp. 9625
Author(s):  
Emma Guitart Font ◽  
Georg A. Sprenger
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Catalytic Efficiency ◽  
Glycerol Dehydrogenase ◽  
Gene Encoding ◽  
E Coli ◽  
Glycerol Formation ◽  
Type Gene ◽  
K 12 ◽  
Glucose 6 Phosphate Dehydrogenase

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h−1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h−1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.

scholarly journals Genetic Evidence for an Interaction of the UbiG O-Methyltransferase with UbiX in Escherichia coli Coenzyme Q Biosynthesis

2006 ◽  
Vol 188 (17) ◽  
pp. 6435-6439 ◽  
Author(s):  
Melissa Gulmezian ◽  
Haitao Zhang ◽  
George T. Javor ◽  
Catherine F. Clarke
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Coenzyme Q ◽  
Genetic Evidence ◽  
Deficient Mutant ◽  
Gene Encoding ◽  
K 12

ABSTRACT IS16 is a thiol-sensitive, Q-deficient mutant strain of Escherichia coli. Here, we show that IS16 harbors a mutation in the ubiG gene encoding a methyltransferase required for two O-methylation steps of Q biosynthesis. Complementation of IS16 with either ubiG or ubiX K-12 reverses this phenotype, suggesting that UbiX may interact with UbiG.

DELETION MAPPING OF zwf, THE GENE FOR A CONSTITUTIVE ENZYME, GLUCOSE 6-PHOSPHATE DEHYDROGENASE IN ESCHERICHIA COLI

Genetics ◽  
1972 ◽  
Vol 71 (4) ◽  
pp. 481-489
Author(s):  
D G Fraenkel ◽  
Santimoy Banerjee
Keyword(s):  
Escherichia Coli ◽  
Fine Structure ◽  
Mutant Strain ◽  
Genetic Map ◽  
Gene Order ◽  
Sugar Metabolism ◽  
Deletion Mapping ◽  
Secondary Mutation ◽  
E Coli ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACT Genes for three enzymes of intermediary sugar metabolism in E. coli, zwf (glucose 6-phosphate dehydrogenase, constitutive), edd (gluconate 6-phosphate dehydrase, inducible), and eda (2-keto-3-deoxygluconate 6-phosphate aldolase, differently inducible) are closely linked on the E. coli genetic map, the overall gene order being man… old… eda. edd. zwf… cheB… uvrC… his. One class of apparent revertants of an eda mutant strain contains a secondary mutation in edd, and some of these mutations are deletions extending into zwf. We have used a series of spontaneous edd-zwf deletions to map a series of point mutants in zwf and thus report the first fine structure map of a gene for a constitutive enzyme (zwf).

scholarly journals mcr-9, an Inducible Gene Encoding an Acquired Phosphoethanolamine Transferase in Escherichia coli, and Its Origin

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Nicolas Kieffer ◽  
Guilhem Royer ◽  
Jean-Winoc Decousser ◽  
Anne-Sophie Bourrel ◽  
Mattia Palmieri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Acquired Resistance ◽  
Regulatory System ◽  
Amino Acid Identity ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
K 12 ◽  
A Minor

ABSTRACT The plasmid-located mcr-9 gene, encoding a putative phosphoethanolamine transferase, was identified in a colistin-resistant human fecal Escherichia coli strain belonging to a very rare phylogroup, the D-ST69-O15:H6 clone. This MCR-9 protein shares 33% to 65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1 to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae. Analysis of the lipopolysaccharide of the MCR-9-producing isolate revealed a function similar to that of MCR-1 by adding a phosphoethanolamine group to lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once the mcr-9 gene was cloned and produced in an E. coli K-12-derived strain. Nevertheless, we showed here that subinhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9. Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate silently and remain undetected unless induced by colistin.

scholarly journals Characterization of Methylglyoxal Synthase fromClostridium acetobutylicum ATCC 824 and Its Use in the Formation of 1,2-Propanediol

1999 ◽  
Vol 65 (7) ◽  
pp. 3244-3247 ◽  
Author(s):  
Ke-xue Huang ◽  
Frederick B. Rudolph ◽  
George N. Bennett
Keyword(s):  
Escherichia Coli ◽  
Clostridium Acetobutylicum ◽  
Glycerol Dehydrogenase ◽  
Dihydroxyacetone Phosphate ◽  
Gene Encoding ◽  
E Coli ◽  
Native Molecular Mass ◽  
Optimum Ph ◽  
Methylglyoxal Synthase

ABSTRACT A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The Km andV max values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min−1μg−1, respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.

scholarly journals Apramycin resistance plasmids inEscherichia coli: possible transfer toSalmonella typhimuriumin calves

1992 ◽  
Vol 108 (2) ◽  
pp. 271-278 ◽  
Author(s):  
J. E. B. Hunter ◽  
J. C. Shelley ◽  
J. R. Walton ◽  
C. A. Hart ◽  
M. Bennett
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
High Frequency ◽  
Inescherichia Coli ◽  
Gene Encoding ◽  
E Coli ◽  
Resistance Plasmids ◽  
Effect Of Treatment ◽  
K 12

SUMMARYAn outbreak of salmonellosis in calves was monitored for persistence ofSalmonella typhimuriumexcretion in faeces and the effect of treatment with apramycin. Prior to treatment apramycin-resistantEscherichia coliwere present but allS. typhimuriumisolates were sensitive. Following the treatment of six calves with apramycin, apramycin-resistantS. typhimuriumwere isolated from two treated calves and one untreated calf. Plasmid profiles ofE. coliandS. typhimuriumwere compared and plasmids conferring resistance to apramycin and several other antibiotics were transferred by conjugationin vitrofrom calfE. coliandS. typhimuriumisolates toE. coliK-12 and fromE. colitoS. typhimurium. The plasmids conjugated with high frequencyin vitrofromE. colitoS. typhimurium, and hybridized to a DNA probe specific for the gene encoding aminoglycoside acetyltransferase 3-IV (AAC(3)-IV) which confers resistance to apramycin, gentamicin, netilmicin and tobramycin.

scholarly journals The Global Regulatory hns Gene Negatively Affects Adhesion to Solid Surfaces by Anaerobically Grown Escherichia coli by Modulating Expression of Flagellar Genes and Lipopolysaccharide Production

2002 ◽  
Vol 184 (6) ◽  
pp. 1522-1529 ◽  
Author(s):  
Paolo Landini ◽  
Alexander J. B. Zehnder
Keyword(s):  
Escherichia Coli ◽  
Sharp Decrease ◽  
Luria Broth ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Aerobic Conditions ◽  
E Coli ◽  
Hydrophilic Surfaces ◽  
K 12 ◽  
Initial Binding

ABSTRACT The initial binding of bacterial cells to a solid surface is a critical and essential step in biofilm formation. In this report we show that stationary-phase cultures of Escherichia coli W3100 (a K-12 strain) can efficiently attach to sand columns when they are grown in Luria broth medium at 28°C in fully aerobic conditions. In contrast, growth in oxygen-limited conditions results in a sharp decrease in adhesion to hydrophilic substrates. We show that the production of lipopolysaccharide (LPS) and of flagella, as well as the transcription of the fliC gene, encoding the major flagellar subunit, increases under oxygen-limited conditions. Inactivation of the global regulatory hns gene counteracts increased production of LPS and flagella in response to anoxia and allows E. coli W3100 to attach to sand columns even when it is grown under oxygen-limited conditions. We propose that increased production of the FliC protein and of LPS in response to oxygen limitation results in the loss of the ability of E. coli W3100 to adhere to hydrophilic surfaces. Indeed, overexpression of the fliC gene results in a decreased adhesion to sand even when W3100 is grown in fully aerobic conditions. Our observations strongly suggest that anoxia is a negative environmental signal for adhesion in E. coli.

scholarly journals Active Genetic Elements Present in the Locus of Enterocyte Effacement in Escherichia coli O26 and Their Role in Mobility

2006 ◽  
Vol 74 (7) ◽  
pp. 4190-4199 ◽  
Author(s):  
Maite Muniesa ◽  
Mark A. Schembri ◽  
Nadja Hauf ◽  
Trinad Chakraborty
Keyword(s):  
Escherichia Coli ◽  
Direct Repeat ◽  
Gene Encoding ◽  
Integrase Gene ◽  
E Coli ◽  
Recombination Processes ◽  
Strong Homology ◽  
Enterocyte Effacement ◽  
K 12 ◽  
Trna Locus

ABSTRACT The locus of enterocyte effacement (LEE) is a large multigene chromosomal segment encoding gene products responsible for the generation of attaching and effacing lesions in many diarrheagenic Escherichia coli strains. A recently sequenced LEE harboring a pathogenicity island (PAI) from a Shiga toxin E. coli serotype O26 strain revealed a LEE PAI (designated LEE O26) almost identical to that obtained from a rabbit-specific enteropathogenic O15:H− strain. LEE O26 comprises 59,540 bp and is inserted at 94 min within the mature pheU tRNA locus. The LEE O26 PAI is flanked by two direct repeats of 137 and 136 bp (DR1 and DR2), as well as a gene encoding an integrase belonging to the P4 integrase family. We examined LEE O26 for horizontal gene transfer. By generating mini-LEE plasmids harboring only DR1 or DR2 with or without the integrase-like gene, we devised a simple assay to examine recombination processes between these sequences. Recombination was shown to be integrase dependent in a ΔrecA E. coli K-12 strain background. Recombinant plasmids harboring a single direct repeat cloned either with or without the LEE O26 integrase gene were found to insert within the chromosomal pheU locus of E. coli K-12 strains with equal efficiency, suggesting that an endogenous P4-like integrase can substitute for this activity. An integrase with strong homology to the LEE O26 integrase was detected on the K-12 chromosome associated with the leuX tRNA locus at 97 min. Strains deleted for this integrase demonstrated a reduction in the insertion frequency of plasmids harboring only the DR into the pheU locus. These results provide strong evidence that LEE-harboring elements are indeed mobile and suggest that closely related integrases present on the chromosome of E. coli strains contribute to the dynamics of PAI mobility.

scholarly journals Sequence Analysis of the GntII (Subsidiary) System for Gluconate Metabolism Reveals a Novel Pathway for l-Idonic Acid Catabolism in Escherichia coli

1998 ◽  
Vol 180 (14) ◽  
pp. 3704-3710 ◽  
Author(s):  
Christoph Bausch ◽  
Norbert Peekhaus ◽  
Cristina Utz ◽  
Tessa Blais ◽  
Elizabeth Murray ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Genomic Sequence ◽  
Regulatory Protein ◽  
Gene Encoding ◽  
E Coli ◽  
Cell Extracts ◽  
Genomic Sequence Analysis ◽  
Metabolic Sequence ◽  
K 12

ABSTRACT The presence of two systems in Escherichia coli for gluconate transport and phosphorylation is puzzling. The main system, GntI, is well characterized, while the subsidiary system, GntII, is poorly understood. Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of l-idonic acid in whichd-gluconate is an intermediate. The genes have been named accordingly: the idnK gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the idnD-idnO-idnT-idnRoperon, which encodes l-idonate 5-dehydrogenase, 5-keto-d-gluconate 5-reductase, an l-idonate transporter, and an l-idonate regulatory protein, respectively. The metabolic sequence is as follows: IdnT allows uptake of l-idonate; IdnD catalyzes a reversible oxidation ofl-idonate to form 5-ketogluconate; IdnO catalyzes a reversible reduction of 5-ketogluconate to formd-gluconate; IdnK catalyzes an ATP-dependent phosphorylation of d-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway; and IdnR appears to act as a positive regulator of the IdnR regulon, withl-idonate or 5-ketogluconate serving as the true inducer of the pathway. The l-idonate 5-dehydrogenase and 5-keto-d-gluconate 5-reductase reactions were characterized both chemically and biochemically by using crude cell extracts, and it was firmly established that these two enzymes allow for the redox-coupled interconversion of l-idonate andd-gluconate via the intermediate 5-ketogluconate. E. coli K-12 strains are able to utilize l-idonate as the sole carbon and energy source, and as predicted, the ability ofidnD, idnK, idnR, andedd mutants to grow on l-idonate is altered.

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